Use of polymerase chain reaction for searching for producers of ergot alkaloids from among microscopic fungi]

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the genera Penicillium and Claviceps was evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerated primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as alpha-cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

An Ergot Alkaloid Biosynthesis Gene and Clustered Hypothetical Genes from Aspergillus fumigatus

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.     

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession numbers are given in the text and in Fig. 3) Dedicated to Dr. Dr. h. c. mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 100th birthday     

An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.     

The Fungus Penicillium citrinum Thom 1910 VKM FW-800 Isolated from Ancient Permafrost Sediments As a Producer of the Ergot Alkaloids Agroclavine-1 and Epoxyagroclavine-1

The study of the secondary metabolites of the relict strain Penicillium citrinum VKM FW-800 isolated from ancient Arctic permafrost sediments showed that this fungus produces agroclavine-1 and epoxyagroclavine-1, which are rare ergot alkaloids with the 5R,10S configuration of the tetracyclic ergoline ring system. The production of the alkaloids by the fungus showed a biphasic behavior, being intense in the phase of active growth and slowing down in the adaptive lag phase and in the stationary growth phase. The addition of zinc ions to the incubation medium led to a fivefold increase in the yield of the alkaloids. The alkaloid-producing Penicillium fungi isolated from different regions exhibited the same tendencies of growth and alkaloid production.     

An Old Yellow Enzyme Gene Controls the Branch Point between Aspergillus fumigatus and Claviceps purpurea Ergot Alkaloid Pathways

Ergot fungi in the genus Claviceps and several related fungal groups in the family Clavicipitaceae produce toxic ergot alkaloids. These fungi produce a variety of ergot alkaloids, including clavines as well as lysergic acid derivatives. Ergot alkaloids are also produced by the distantly related, opportunistic human pathogen Aspergillus fumigatus. However, this fungus produces festuclavine and fumigaclavines A, B, and C, which collectively differ from clavines of clavicipitaceous fungi in saturation of the last assembled of four rings in the ergoline ring structure. The two lineages are hypothesized to share early steps of the ergot alkaloid pathway before diverging at some point after the synthesis of the tricyclic intermediate chanoclavine-I. Disruption of easA, a gene predicted to encode a flavin-dependent oxidoreductase of the old yellow enzyme class, in A. fumigatus led to accumulation of chanoclavine-I and chanoclavine-I-aldehyde. Complementation of the A. fumigatus easA mutant with a wild-type allele from the same fungus restored the wild-type profile of ergot alkaloids. These data demonstrate that the product of A. fumigatus easA is required for incorporation of chanoclavine-I-aldehyde into more-complex ergot alkaloids, presumably by reducing the double bond conjugated to the aldehyde group, thus facilitating ring closure. Augmentation of the A. fumigatus easA mutant with a homologue of easA from Claviceps purpurea resulted in accumulation of ergot alkaloids typical of clavicipitaceous fungi (agroclavine, setoclavine, and its diastereoisomer isosetoclavine). These data indicate that functional differences in the easA-encoded old yellow enzymes of A. fumigatus and C. purpurea result in divergence of their respective ergot alkaloid pathways.Different classes of ergot alkaloids are produced by members of two distinct fungal lineages. Clavicipitaceous species, which include Claviceps spp. and Neotyphodium spp., are in the order Hypocreales and typically synthesize lysergic acid derivatives (13, 16, 18). These alkaloids have a double bond in the last assembled of four rings (D ring) of the tetracyclic ergoline ring structure. Ergot alkaloids are also produced by the distantly related opportunistic human pathogen Aspergillus fumigatus, a member of the order Eurotiales (8, 14, 16, 18). Ergot alkaloids of A. fumigatus are of the clavine class and differ from the more complex profile of Claviceps purpurea and Neotyphodium spp. One important distinction between the ergot alkaloids produced by these different fungi is the saturation of the fourth ring of the ergoline structure in A. fumigatus (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Structures and relationships of relevant ergot alkaloids. (A) Chanoclavine-I is oxidized to its aldehyde form before being incorporated into festuclavine (and downstream alkaloids) in A. fumigatus or agroclavine (and downstream alkaloids) in C. purpurea. (B) Conventional ring labeling and atom numbering referred to in the text.Several genes involved in the ergot alkaloid pathways of A. fumigatus and clavicipitaceous fungi are found clustered together in the genome of each species (3, 4, 6, 18, 23). These distantly related fungi are hypothesized to share several early pathway steps, after which the pathways diverge to yield distinct sets of ergot alkaloids (3, 13, 16). The gene dmaW, which encodes dimethylallyltryptophan (DMAT) synthase, catalyzes the prenylation of tryptophan that initiates the ergot alkaloid pathway in clavicipitaceous fungi (22, 25) and functions similarly in A. fumigatus (3, 24). The region surrounding this gene in A. fumigatus contains homologues of genes also found in Neotyphodium lolii and C. purpurea ergot alkaloid gene clusters (3, 4, 6). One of the shared genes, easA, is predicted to encode a member of the old yellow enzyme (OYE) family of oxidoreductases. Old yellow enzymes are flavin-containing oxidoreductases initially found in the brewer''s bottom yeast Saccharomyces carlsbergensis (26). Enzymes in this family use a reduced flavin cofactor and an active-site tyrosine residue to reduce the carbon-carbon double bond in an α/β-unsaturated aldehyde or ketone (7, 10). Subsequently, the enzymes require NADPH to restore the flavin cofactor to its reduced state. OYEs catalyze multiple reactions useful for both biotechnological and pharmaceutical applications; however, physiological roles and natural substrates for many of these enzymes presently are unknown (26). On the basis of the apparent need in the ergot alkaloid pathway of A. fumigatus for reduction of a carbon-carbon double bond in the intermediate chanoclavine-I-aldehyde, we hypothesized that the OYE-encoding gene easA is required for ergot alkaloid biosynthesis (3, 16). In this study, easA in A. fumigatus was disrupted and complemented to ascertain the role of its gene product in ergot alkaloid biosynthesis.     

Ergoline alkaloids in convolvulaceous host plants originate from epibiotic clavicipitaceous fungi of the genus Periglandula

Ergoline (i.e., ergot) alkaloids are a group of physiologically active natural products occurring in the taxonomically unrelated fungal and plant taxa, Clavicipitaceae and Convolvulaceae, respectively. The disjointed occurrence of ergoline alkaloids seems to contradict the frequent observation that identical or at least structurally related natural products occur in organisms with a common evolutionary history. This problem has now been solved by the finding that not only graminaceous but also some dicotyledonous plants belonging to the family Convolvulaceae, such as Ipomoea asarifolia and Turbina corymbosa, form close associations with ergoline alkaloid producing fungi, Periglandula ipomoeae and Periglandula turbinae. These species belong to the newly established genus Periglandula within the Clavicipitaceae. The fungus–plant associations are likely to be mutualistic symbioses.     

Specificity in the interaction between an epibiotic clavicipitalean fungus and its convolvulaceous host in a fungus/plant symbiotum

Ipomoea asarifolia and Turbina corymbosa (Convolvulaceae) are associated with epibiotic clavicipitalean fungi responsible for the presence of ergoline alkaloids in these plants. Experimentally generated plants devoid of these fungi were inoculated with different epibiotic and endophytic fungi resulting in a necrotic or commensal situation. A symbiotum of host plant and its respective fungus was best established by integration of the fungus into the morphological differentiation of the host plant. This led us to suppose that secretory glands on the leaf surface of the host plant may play an essential role in ergoline alkaloid biosynthesis which takes place in the epibiotic fungus.Key words: ergoline alkaloids, ipomoea, turbina, convolvulaceae, claviceps, balansia, clavicipitaceae, penicillium, plant-fungus symbiotum     

Studies on Sphacelia sorghi McRae, an ergot of Sorghum vulgare Pers.

The constituent ergoline alkaloids produced in vitro by a Nigerian strain of Sphacelia sorghi have been identified as dihydroergosine, festuclavine, pyroclavine, dihydroelymoclavine, and chanoclavine. The same alkaloids were present in both the sphacelial stage and the sclerotia when S. sorghi parasitized florets of Sorghum vulgare. The Nigerian fungus appears to be quite different from certain oriental collections bearing the same name, and although forming stromatal initials, failed to develop the sexual stage. Mice successfully raised litters and showed no adverse response when fed on diets containing up to 50% of the ergot sclerotia. The ergot had also no effect on early pregnancy. Some of the alkaloid was excreted in the faeces, but, as injected alkaloid was also shown to be excreted in the faeces, this could have represented alkaloid which had been absorbed from the ingested sclerotia and re-excreted via the bile.     

Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.     

Effect of microelements on the biosynthesis of secondary metabolites by the fungusPenicillium citrinum thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondaryO-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions slightly affected fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

Physiological status of submergedClaviceps during enzymic assembly of ergot alkaloids

Ergot alkaloids are formed only for arelatively brief time during the lifespan of the culture and under conditions of reduced proliferation. They cannot be taken for waste products of general metabolism. Ergot strains are capable of carrying out all the simple steps of the anthranilic acid—tryptophan cycles. Alkaloids influence activities of certain enzymes of primary metabolism in the ergot mycelium,e.g. tryptophan synthetase, acetyl-CoA carboxylase, citrate synthase, isocitrate lyase, and malate synthase. Ergot alkaloids do not belong to a group of physiologically inert secondary metabolites. A tentative scheme of the enzymic assembly of the ergoline nucleus is presented. The increased yield of ergoline alkaloids may be attributed to the following points: (1) Unbalnced growth of the culture. (2) Support of competition of fatty acids and alkaloid biosynthesis for acetyl-CoA. (3) Decreased activities of tricarboxylic acid and glyoxylate cycles. (4) Positive association between the rate of protein turnover and alkaloid formation. (5) Stimulation of both tryptophan synthesis and degradation via kynurenine—anthranilate. (6) Regulation of tryptophan-histidine cross-pathway. (7) Continuous control of the alkaloid level during fermentation.     

The fungus Penicillium citrinum Thom 1910 VKM FW-800 isolated from ancient permafrost sediments as a producer of the ergot alkaloids agroclavine-1 and epoxyagroclavine-1

The study of the secondary metabolites of the relict strain Penicillium citrinum VKM FW-800 isolated from ancient Arctic permafrost sediments showed that this fungus produces agroclavine-1 and epoxyagroclavine-1, which are rare ergot alkaloids with the 5R,10S configuration of the tetracyclic ergoline ring system. The production of the alkaloids by the fungus showed a biphasic behavior, being intense in the phase of active growth and slowing down in the adaptive lag phase and in the stationary growth phase. The addition of zinc ions to the incubation medium led to a fivefold increase in the yield of the alkaloids. The alkaloids-producing Penicillium fungi isolated from different regions exhibited the same tendencies of growth and alkaloid production.     

Relationship between the Claviceps Life Cycle and Productivity of Ergot Alkaloids

Abstract

The morphology, biochemistry, and physiology studies during development of Claviceps purpurea fungi clearly demonstrate that alkaloid synthesis is linked to a specific stage of the fungal life cycle. In nature, ergot alkaloids are synthesized in the course of developing sclerotia, while in submerged cultures, lacking sexual reproduction, alkaloid synthesis proceeds in sclerotia-like cells. Highly active submerged strains could be obtained by combination of mutagens with a different mode of action as well as by somatic hyphal anastomoses or efficient protoplast fusions to obtain the parasexual cycle. Fused strains not only retained the biosynthetic activity of parent strains but produced even much higher amounts of alkaloids. In our strains, the appropriate morphology always corresponded to high productivity. Furthermore, the form of cell differentiation was typical for each particular strain. When comparing active and inactive strains, measurements of qualitative and quantitative changes in mycelium composition revealed different metabolic patterns and certain characteristics necessary for efficient alkaloid production. Evaluation of activities of some enzymes from the central metabolic pathways, which generate the basic intermediates for ergot alkaloid synthesis also contributed to the overall knowledge of mechanisms involved.     

Development of a UHPLC-FLD method for the analysis of ergot alkaloids and application to different types of cereals from Luxembourg

Ergot alkaloids are toxins produced by some species of fungi in the genus Claviceps, that may infect rye and triticale and, in a minor degree, other types of cereals. In this study, a new UHPLC-FLD method for the quantification of the six major ergot alkaloids as well as their corresponding epimers was developed. The sample preparation was done by a solid-liquid extraction with acetonitrile and clean-up via freeze-out. The method was fully validated and then applied to 39 samples (wheat, rye, triticale, and barley) harvested in Luxembourg in 2016. Samples were sieved (1.9?×?20 mm) prior to analysis in order to remove sclerotia, hosting the alkaloids. However, 23 samples still contained at least one ergot alkaloid >?LOQ and concentrations of the sum of the 6 ergot alkaloids ranged from 0.3 to 2530.1 μg/kg. Interestingly, the highest concentrations were measured in wheat and not in rye or triticale, suggesting that all kinds of cereals should be included in monitoring programs. The outcome of this study allowed giving a first overview of ergot alkaloid concentrations in cereals harvested in Luxembourg, and the measured concentrations were in similar ranges than in other parts of the world (e.g., Canada, France, Germany).     

Biosynthesis of ergot alkaloids. Some new results on an old problem (Review)]

The biosynthetic pathway leading from L-tryptophan, mevalonic acid and methionine to the tetracyclic ergoline ring system of the ergot alkaloids in Claviceps species is reviewed. This pathway entails many mechanistically intriguing features. Recent studies are also discussed which reveal the stereochemical course of the isoprenylation of tryptophan and of the N-methylation of dimethylallyltryptophan (DMAT) and which shed some light on the likely steps leading from the open-chain precursors, N-methyl-DMAT to the tricyclic intermediate, chanoclavine-1. Finally, some plans are outlined to probe the evolutionary relationship of ergot alkaloid biosynthesis in fungi to that in higher plants of the family Convolvulaceae.     

Lumi-ergometrine – structural identification and occurrence in sclerotia

The fungus Claviceps purpurea grows on grasses and cereal grains and produces six predominant ergot alkaloids. These toxic substances undergo different transformation reactions during storage and cereal processing. One of these reactions is the addition of water to a double bond in the ergoline skeleton. Since light is required for this process, the substances formed were named lumi-ergot alkaloids. From these, a new asymmetric carbon and consequently two epimers with different polarities are formed. For investigations of lumi-ergot alkaloids, ergometrine was used exemplarily as it represents one of the six ergot alkaloids predominantly formed by Claviceps purpurea. The main reaction product, the less polar compound of the two lumi-ergometrine epimers, was separated by HPLC and unambiguously identified as 10-(S)-lumi-ergometrine using X-ray structural analysis. A HPLC-MS/MS method was developed for the detection of this substance in sclerotia extracts. Using this method, the existence of both epimeric forms of lumi-ergometrine could be proved in the sclerotia. This is the first time that the existence of a lumi-transformation product of ergot alkaloids was proved in naturally grown samples.     

Biotechnology and genetics of ergot alkaloids

Ergot alkaloids, i.e. ergoline-derived toxic metabolites, are produced by a wide range of fungi, predominantly by members of the grass-parasitizing family of the Clavicipitaceae. Naturally occurring alkaloids like the D-lysergic acid amides, produced by the "ergot fungus" Claviceps purpurea, have been used as medicinal agents for a long time. The pharmacological effects of the various ergot alkaloids and their derivatives are due to the structural similarity of the tetracyclic ring system to neurotransmitters such as noradrenaline, dopamine or serotonin. In addition to "classical" indications, e.g. migraine or blood pressure regulation, there is a wide spectrum of potential new applications of this interesting group of compounds. The biotechnology of ergot alkaloids has a long tradition, and efficient parasitic and submerse production processes have been developed; the biochemistry of the pathway and the physiology of production have been worked out in detail. The recent identification of a cluster of genes involved in ergot alkaloid biosynthesis in C. purpurea and the availability of molecular genetic techniques allow the development of strategies for rational drug design of ergoline-related drugs by enzyme engineering and by biocombinatorial approaches.     

Ergot alkaloids: Quantitation and recognition challenges

Bread, flour, infant formula and baby food samples (n=109, from which n=54 made of or containing rye), collected in 2001, 2003, and 2005, were analysed for ergot alkaloids. Samples were extracted using acidic conditions and the extracts subjected to an automated solid-phase clean up using combined cation exchange/reversed-phase sorbent cartridges (Oasis-MCX). Subsequent chromatographic separation and analysis was performed by liquid chromatography (LC) with fluorescence detection (FLD) and by LC with mass spectrometric detection (MS/MS). The ergot alkaloid (EAs) content of a sample was defined as the sum of the 16 alkaloids ergometrin(in)e, ergosin(in)e, ergotamin(in)e, ergostin(in)e, ergocornin(in)e, α-ergocryptin(in)e, β-ergocryptin(in)e and ergocristin(in)e. Comparability of results obtained by LC-FLD and LC-MS/MS was satisfactory, but varied for different alkaloids. The use of dihydro-ergocristine as an internal standard considerably improved the reliability of analytical data from LC-MS/MS. Compared with earlier data (Baumannet al., 1985) for median levels of ergot alkaloids in rye flour (140 ng/g) and bread (21.3 ng/g) from Switzerland, the median values for ergot alkaloids in rye flour collected in 2001 (n=13) and in 2005 (n=2) were 172 ng/g and 160 ng/g, respectively. The median values for bread (fresh weight) collected in 2001 (n=14), 2003 (n=7), and 2005 (n=2) were 87 ng/g, 120 ng/g, and 156 ng/g, respectively. Low levels of ergot alkaloids were also found in wheat products and in some infant formulae and baby foods containing rye. By additional LC-MS/MS experiments, the possible natural occurrence of ergot congeners containing the 9,10-unsaturated ergoline cation (m/z=223) was investigated. In a few samples, ergovalin(in)e was tentatively identified by these means. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007     

Contribution of ergot alkaloids to suppression of a grass-feeding caterpillar assessed with gene knockout endophytes in perennial ryegrass

Neotyphodium and Epichloë species (Ascomycota: Clavicipitaceae) are fungal symbionts (endophytes) of grasses. Many of these endophytes produce alkaloids that enhance their hosts’ resistance to insects or are toxic to grazing mammals. The goals of eliminating from forage grasses factors such as ergot alkaloids that are responsible for livestock disorders, while retaining pasture sustainability, and of developing resistant turf grasses, require better understanding of how particular alkaloids affect insect herbivores. We used perennial ryegrass Lolium perenne L. (Poaceae) symbiotic with Neotyphodium lolii × Epichloë typhina isolate Lp1 (a natural interspecific hybrid), as well as with genetically modified strains of Lp1 with altered ergot alkaloid profiles, to test effects of ergot alkaloids on feeding, growth, and survival of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), a generalist grass‐feeding caterpillar. Neonates or late instars were provided clippings from glasshouse‐grown plants in choice and rearing trials. Wild‐type endophytic grass showed strong antixenosis and antibiosis, especially to neonates. Plant‐endophyte symbiota from which complex ergot alkaloids (ergovaline and lysergic acid amides such as ergine) or all ergot alkaloids were eliminated by endophyte gene knockout retained significant resistance against neonates. However, this activity was reduced compared to that of wild‐type Lp1, providing the first direct genetic evidence that ergot alkaloids contribute to insect resistance of endophytic grasses. Similarity of larval response to the two mutants suggested that ergovaline and/or ergine account for the somewhat greater potency of wild‐type Lp1 compared to the knockouts, whereas simpler ergot alkaloids contribute little to that added resistance. All of the endophyte strains also produced peramine, which was probably their primary resistance component. This study suggests that ergot alkaloids can be eliminated from an endophyte of perennial ryegrass while retaining significant insect resistance.